Streptococcus pyogenes, also known as group A streptococcus (GAS), is a major human pathogen that causes significant morbidity and mortality. GAS infections can lead to several disease conditions including rheumatic heart disease (RHD), the major cause of acquired heart disease in children. Globally, at least 34 million people living with RHD causing 345,000 deaths per year. Thus, the development of a human GAS vaccine remains a healthcare priority. However, a broadly protective licensed GAS vaccine remains elusive due to the antigenic variation in vaccine candidates among different GAS serotypes, genetic diversity of the pathogen, and cross-reactivity of antibodies against GAS antigens with human tissues. To overcome these challenges and protect public health from GAS diseases, it is critical to identify novel vaccine targets and/or develop new vaccination strategies that produce broad and effective protection against GAS diseases. Our recent studies demonstrated that the highly conserved bacterial metal acquisition systems are critical virulence determinants and effective vaccine targets capable of conferring cross-serotypic protection against GAS diseases. The metal importers compete with host nutritional immune mechanisms to acquire metals during infection and promote bacterial survival in hostile host environments. Host deploys nutritional immune mechanisms, as components of innate immunity, to retard microbial growth by nutrient deprivation. GAS infected abscesses are enriched with host factor, calprotectin (CP), which sequesters Zn from the colonization surfaces to limit GAS growth. However, GAS withstands CP onslaught and successfully replicates in the host by employing the high-affinity Zn importer, AdcABC. A major caveat to this model is that, in GAS and other gram-positive bacteria, the cell membrane-bound AdcABC-like importers are buried underneath the thick cell wall layer. The masked subcellular localization of AdcABC fails to explain its function as a competitive Zn uptake mechanism against the efficient host nutritional defenses and its efficacy as a vaccine target. The primary objective of this proposal is to determine the mechanisms by which GAS uses AdcA to evade host nutritional defenses, and evaluate novel AdcA- based vaccination strategies for its protective efficacy against human-like GAS infections. Using a multidisciplinary approach, we will test the central hypothesis of this proposal that GAS uses non- replicating, cell-free membrane vesicles (MV) coated with AdcABC for Zn acquisition and subverts CP- mediated Zn limitation. At the completion of the proposed study, the mechanistic basis for MV-mediated GAS Zn acquisition will be delineated and protective efficacy of protein- and MV-based AdcA vaccination for GAS disease prevention will be assessed.